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Fungal symbiotic associations
of the ambrosia beetle
Euwallacea nr. fornicatus
Luisa Cruz and Daniel Carrillo
IFAS-Tropical Research and Education Center, University of Florida, Homestead
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Introduction
• Euwallacea fornicatus a complex of cryptic species attacking avocado.
• Fungal-farmers, presence of mycangia for fungal transport.
• E. fornicatus primary symbionts are members of the Ambrosia Fusarium Clade (AF-1 to AF- 12), Graphiumspp. and Acremonium spp.
• Native to South Asia (Established in Israel, Australia and South Africa, Central and North America)
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FloridaTea Shot-hole Borer (TSHB)
Euwallacea nr. fornicatus
2002 Royal Poinciana
2012 First Avocado tree
2013-15 10 avocado groves
2016 First outbreak (1500 trees in a grove)
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• TSHB Attacks and kills medium and small branches• Interior-shaded braches first, later outer branches• Base of the branch, later all the branch
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• Branch dieback • May limit production
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Fusarium sp. AF-8 AF-6
Graphium sp.Acremonium sp. A. morum
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ObjectiveTo understand the symbiotic relationship of E. fornicatus and its fungal associates:
• I) Survival and development of larvae on individual cultures of the symbionts
• II) Rearing on avocado sawdust media inoculated with the individual symbionts
• III) Production of lines carrying a single Fusarium spp.
• IV) Infesting avocado trees with the monosymbiotic lines
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Fusarium sp. AF-8 AF-6
Graphium sp.Acremonium sp. A. morum
• E. fornicatus were excavated from infested avocado logs (Miami- Dade County Florida USA)
• Fungal isolates were obtained from sapwood tissue and beetles mycangia
Beetles collection and rearing conditions and fungal strains
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Fusarium sp. nov. AF-8 AF-6
Graphium sp. Acremonium sp. A. morum
I) Survival and development of E. fornicatus larvae on symbiont cultures
• Third instar larvae were collected and surface disinfected
• Symbiont cultures on PDA media were used as the larvae rearing substrate. R. arxii was used as a control
• Larva survival, pupation and emergence of adults were recorded
Raffaelea arxii
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I ) Development of E. fornicatus larvae on symbiont cultures
* **
*
**
*
*
*
* *
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• Beetles can develop in a single symbiont but probably need more than one symbiont to develop strong colonies.
• Fusarium sp. and AF-8 important food for immature development
• AF-6, Graphium sp. and Acremonium morum important for adult stage.
I ) Development of E. fornicatus larvae on symbiont cultures
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Fusarium sp.
AF-8
AF-6
Graphium sp.
Acremonium sp.
A. morum
II) Rearing of E. fornicatus on media inoculated with individual symbionts
• Media inoculated with (1*106 CFU) a given symbiont
• Colony dissection 40 days• The number of
developemental stages was recorded
• Beetles symbionts were isolated and ID
• The experiment was carried out for two generations
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II) Effect of media inoculation on the reproduction of E. fornicatus
Total Brood
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ab
bc
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aba
abc
Females
a
abb
b
aa
ab
*
**
*
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II) Beetle fungal associates
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• Fusarium spp. tend to colonize the mycangia of the beetles more than other symbionts.
• AF-6 was the symbiont with the lowest frequency in the mycangia.
• Reproduction was lower in the media inoculated with AF-8 and AF-6.
II) Beetle fungal associates
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III) Production of E. fornicatus lines monosymbiontic colonies
Fusarium sp. nov. AF-8 AF-6
• Pupae were surface disinfected
• Reared on Fusariumspp. cultures
• Mature fertilized females were transferred to rearing tubes
• Offspring females were evaluated for the presence of a single Fusarium sp.
PBS tween2% Bleach 30sec
H2O70% Et-OH30 sec
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a
b
a
aa
aa
b
a
a
a
aa
b
b
a
III) E. fornicatus lines monosymbiontic lines reproduction
Females Total Brood
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• Fusarium sp. and AF-8 were the best nutritional symbionts comparable to the control.
III) E. fornicatus lines monosymbiontic lines reproduction
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• Five beetles per monosymbiotic line and a lab colony
• Treatments were distributed in independent branches of the same tree
• Evaluation 50 days after infestation *Beetles activity (boring, reproduction)*Presence of the fungal pathogen
IV) Tree infestation with the E. fornicatus monosymbiotic lines
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Monosymbiotic line Nº infected
trees
Xylem staining
length (range) Fusarium sp. 7/20 8.4 cm (2-17 cm)
AF-8 10/20 5.9cm (2-11 cm)AF-6 3/20 4.5 cm (6-6 cm)
Control 7/20 8.5 cm (5-11 cm)
IV) Tree infestation with the E. fornicatus monosymbiotic colonies
Reproduction was not accomplished for any of the tree monosymbiotic lines nor the control laboratory colony
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Conclusions • Fusarium sp. and AF-8 appear to be the most important nutritional
symbionts and the strongest pathogen.
• AF-6 seems to be a poor nutritional symbiont and a weak pathogen.
Future research
• Pathogenicity of symbionts
• Influence of symbionts on the beetle’s behavior.
• Can fungal volatiles improve current attractants
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¡Gracias!
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II) Fungal associates
Beetles isolates
Galleries isolates
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II) Effect of media inoculation on the reproduction of E. fornicatus
bbc
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iduals
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Females Total Brood